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Anther development, meiosis and pollen formation in Zea tassels cultured in defined liquid medium
Plant Science Letters, 26 (1982) 139--145 Elsevier Scientific Publishers Ireland Ltd.
139
ANTHER DEVELOPMENT, MEIOSIS AND POLLEN FORMATION IN ZEA TASSELS CULTURED IN DEFINED LIQUID MEDIUM
P.L. P O L O W I C K
and R.I. G R E Y S O N
Department of Plant Sciences, Universityof Western Ontario, London, Ontario N 6 A 5B7 (Canada)
(Received October 12th, 1981) (Revision received December 15th, 1981) (Accepted January 13th, 1982)
SUMMARY
Immature tassels of corn (Zea mays L.), excised at a stage when the most mature spikelets on the main axis, possessing rudimentary stamen and gynoecial primordia, continue to grow and differentiate when cultured in a defined liquid medium. The fresh weight of the inflorescence increases up to 1000× during the 14
INTRODUCTION
Corn and tassel spikelets are initially bisexual but become unisexual through selective organ abortion [ 1 ]. It is assumed that the regulation of this differentiation involves plant growth substances [2]; however, the validity of this assumption remains unproven. Previous attempts to examine the roles of plant growth regulators in maize sexuality have involved the estimation of the endogenous levels of auxins and gibberellins [3,4] and their exogenous application [5--8]. The results, however, are contradictory and inconclusive. Floral meristems are seldom used for organogenic culture studies [9], and the completion of development, culminating in gamete maturation, is reported in few of the attempted culture studies [ 10--13 ]. Cereals, especially, have Abbreviations: FPA, formalin/propionic acid/alcohol; PMC, pollen mother cell. 0304--4211/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd.
140 been difficult to study [ 14]. The use of in vitro techniques could, potentially, aid in defining the roles of specific molecules in organ differentiation and the triggering of meiosis. As part of a broad study of sexuality in Zea mays, we have established a suitable basal medium [15] and have begun to study the effects of plant growth regulators in the liquid culture of tassels. We find that cytokinins are particularly active in tassel spikelet maturation. The observations presented in this preliminary report relate to b o t h the biochemistry of meiosis and organ differentiation. METHODS AND MATERIALS Plants of Zea m a y s (cv. Seneca-60) are raised in the greenhouse until the 8th leaf emerges and the basal internodes are elongating (approx. 25--30 days from germination). The shoot portions are removed from the pot. The t w o o u t e r m o s t leaves and the remaining exposed leaf-blades are removed to produce 10 cm stem/leaf-sheath cylinders which enclose the immature tassels. The cylinders are surface-sterilized in 15% 'Javex', sodium hypochlorite bleach, for 20 min and rinsed repeatedly in sterile water. The tassels are dissected aseptically from the stems. These tassel explants are approx. 1 cm long and bear more than 600 spikelet primordia, the most advanced of which are located in the mid-region of the central axis. The upper floret of each of these most-mature spikelets possesses b o t h stamens and gynoecium primordia, as yet undifferentiated and made up essentially of non-vacuolate meristematic cells (Fig. 1). Tassels are placed singly into 125 ml Erlenmeyer flasks which contain 40 ml of medium. The basal liquid medium, as reported previously [15], is composed of Murashige and Skoog [ 16 ] mineral salts, White's [ 17 ] vitamins and glycine, myo-inositol (Calbiochem No. 4071; 100 mg/1) and 0.1 M sucrose. Kinetin is added as indicated in the text. The pH is adjusted to 5.8 with NaOH or HC1. The medium is distributed into Erlenmeyer flasks and autoclaved. Cultures are incubated in a growth chamber on a rotary shaker (90 rev./ min) and provided with an 18-h (24°C)/6-h (20°C) photoperiod. The illumination (50 + 10 microeinsteins M -2 s -1 PAR) is provided by cool white fluorescent tubes plus incandescent bulbs. For analysis, cultures are terminated at intervals of up to 14 and occasionally 20 days. For sectioning, the cultured materials are fixed in a modified paraformaldehyde/glutaraldehyde fixative [ 18 ] or formalin/propionic acid/ alcohol (FPA) and e m b e d d e d in paraffin. For c h r o m o s o m e squashes, anthers are fixed in alcohol/glacial acetic acid (3 : 1) and stained with propiocarmine. This report summarizes observations made during an 8-month period, from over 200 cultured tassels. RESULTS AND DISCUSSION In basal medium (control), the 1-cm tassels can elongate up to 25 cm in
141
gl
O Fig. 1. Longitudinal section of a single corn floret at the time of culture showing (a) a n t h e r initials, (g) gynoecium, (gl) glume and (l) lemma. Calibration bar = 50 urn.
length and increase in fresh weight to 1500 mg from the initial 10 mg, in 14 days. Growth is evident within 36 h. A few normally-shaped and sized spikelets (~1 cm at 14 days) develop. The glumes on these spikelets are green, hairy and papery in texture, exhibit prominent venation and enclose the regular complement of lemmas, paleae and lodicules. Six 4-1oculed anthers mature, 3 for each of the t w o florets in the spikelet. Microsporogenesis proceeds to the uninucleate microspore stage. This 'normal' spikelet development is rare; a mean number of 5 spikelets developed on less than 5% o f the tassels cultured in basal medium (Fig. 2, Control}. The frequency and extent of 'normal' spikelet development is enhanced by the addition of kinetin to the medium. Kinetin is effective over a range of concentrations, 10-9--10 -~ M and is most influential at a concentration of 10 -7 M (Fig. 2). The percentage of tassels possessing 'normal' spikelets approaches 80% at this concentration and the mean number of such spikelets per tassel is nearly 18. In the most successful culture to date, a single inflor-
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escence produced 48 'normal' spikelets. This represents the development of 3--5% of the 600 ÷ initials maturing in vivo. The results are variable but t h e trend is consistent. Neither the fresh weight nor the length of the cultured tassels is enhanced by the kinetin. Similar studies with other cytokinins are in progress. There is considerable stamen growth and differentiation during the culture period. Beginning as an undifferentiated primordium (Fig. 1), the stamen matures into a 4-1oculed anther (Fig. 3) attached to a vascuiarized filament. The anthers reach 5 mm in length after 14 days, while comparable in vivo specimens are approx. 6 mm. Anatomically, cultured material resembles the in vivo-grown material with the anther wall consisting of the usual epidermal, endodermal and tapetal layers and, occasionally, a middle layer. Of particular interest is the observation that meiosis takes place in those spikelets which develop normally on cultured tassels, especially in the presence of kinetin. Vasil [19] also noted an association of kinetin with the completion of meiosis in cultured anthers of Allium cepa. Apparently, ours is the first report of the completion of microsporogenesis in a cereal inflorescence cultured when the most mature anthers are at an early initial stage. This stage is at least 3 days prior to meiosis. Undissected tassels of the same cohort of plants continue to develop in vivo for 3--5 days before pollen mother cells (PMC's) are detected. While the detailed analysis of meiosis in culture remains incomplete, it is obvious that many aspects parallel in vivo development. Within 7 days of culture, PMC's and early stages of meiotic prophase I are observed in propiocarmine squashes of anthers, as illustrated by the leptonema-zygonema stage in Fig. 4. Quartets (Fig. 5) and isolated microspores are prevalent after 10 days of culture although a range of stages is observed from individual stamens. Binucleate pollen grains (Fig. 6), bearing distinct though somewhat abbreviated walls and possessing an aperture with thickened margins, are found after 14 days of culture. Similar grains are observed after 10 days of continued growth in vivo. At this stage, the two nuclei are visibly distinct; the vegetative nucleus lies near the aperture while the generative nucleus is attached to the intine, opposite the aperture. Mature pollen, which was observed in several 20~lay-old cultures, contained a dense cytoplasm, presumably because of starch deposition. On occasion, however, stages of the mitotic divisions of the generative nucleus and the resulting trinucleate pollen grains were noted. Despite the consistency of our results with kinetin it is of concern that less than 10% of the spikelets on an inflorescence respond. Perhaps only a certain fraction is sensitive at the time of explanting. The most sensitive fraction of spikelets does not appear to be the most mature in the developmental gradient along the main and branch axes of the tassel, as the 'normal' spikelets are scattered, apparently at random along these axes. Also, the number of developing spikelets is not augmented when slightly older inflorescences are placed into culture. In view of previous experience with grass
144
Fig. 3. T r a n s v e r s e s e c t i o n o f a n a n t h e r a f t e r 10 d a y s o f c u l t u r e in m e d i u m c o n t a i n i n g 10-7 M k i n e t i n . I n d i v i d u a l locules e x h i b i t d i f f e r e n t stages o f m i c r o s p o r o g e n e s i s . Calibrat i o n bar = 50 u m . Fig. 4. Pollen m o t h e r cells at l e p t o n e m a - z y g o n e m a o f Meiosis I a f t e r 7 days of c u l t u r e in m e d i u m c o n t a i n i n g 10-7 M k i n e t i n . C a l i b r a t i o n b a r = 20 ~ m , Fig. 5. Q u a r t e t o f m i c r o s p o r e s a f t e r 10 days o f c u l t u r e in m e d i u m c o n t a i n i n g 10-7 M k i n e t i n . C a l i b r a t i o n b a r = 20 ~m. Fig. 6. B i n u c l e a t e p o l l e n grain a f t e r 14 days o f c u l t u r e in m e d i u m c o n t a i n i n g 10 -7 M k i n e t i n (a = a p e r t u r e ) . C a l i b r a t i o n b a r = 20 ~m.
145 tissues, a n y d e v e l o p m e n t in c u l t u r e is surprising, especially w i t h such a relatively simple m e d i u m . T h e s e reservations regarding n u m b e r s aside, t h e e x t e n t o f d e v e l o p m e n t f r o m s t a m e n initials t o b i n u c l e a t e pollen in 14 d a y s is striking. T h e techn i q u e s h o u l d p r o v e valuable f o r basic c y t o g e n e t i c and m o r p h o l o g i c a l studies. E x p l o r a t i o n s i n t o t h e b i o c h e m i s t r y o f meiosis and a n t h e r - d e v e l o p m e n t w o u l d be facilitated b y t h e i n c o r p o r a t i o n o f labelled-precursors, an a p p r o a c h n o t feasible in t h e i n t a c t c o r n plant. ACKNOWLEDGEMENTS We t h a n k Dr. K. R a m a n f o r his help w i t h t h e early stages o f this w o r k and gratefully a c k n o w l e d g e t h e s u p p o r t o f t h e N a t u r a l Sciences and Engineering Research Council o f C a n a d a f o r a P o s t g r a d u a t e Scholarship t o PLP a n d annual grants t o R I G . We are also grateful t o t h e University o f Western O n t a r i o A c a d e m i c D e v e l o p m e n t F u n d f o r generous s u p p o r t . REFERENCES
1 0 . T . Bonnett, Ann. Mo. Bot. Gard., 35 (1948) 269. 2 J. Heslop-Harrison, Physiology of development: from seeds to sexuality, Vol. VI C, Academic Press, 1972, p. 133. 3 Z. Sladky, Biol. Plant., 11 (1969) 208. 4 S.B. Rood, R.P. Pharis and D.J. Major, Plant Physiol., 66 (1980) 793. 5 P.M. Nelson and E.C. Rossman, Science, 127 (1958) 1500. 6 D.J. Hansen, S.K. Bellman and R.M. Sachet, Crop Sci., 16 (1976) 371. 7 H.N. Krishnamoorthy and A.R. Talukdar, Z. Pflanzenphysiol., 79 (1976) 91. 8 N.H. Nickerson, Ann. Mo. Bot. Gard., 46 (1959) 19. 9 G.S. Hicks, Bot. Rev., 46 (1980) 1. 10 J. Blake, J. Exp. Bot., 20 (1969) 113. 11 D. Porath and E. Galun, Ann. Bot. (London), 31 (1967) 283. 12 G.S. Hicks and I.M. Sussex, Can. J. Bot., 48 (1970) 133. 13 J. Berghoef and J. Bruinsma, Z. Pflanzenphysiol., 93 (1979) 345. 14 C.E. Green, In vitro plant regeneration in cereals and grasses, in: T.A. Thorpe (Ed.), Frontiers of Plant Tissue Culture 1978, International Assoc. for Plant Tissue Culture, Calgary, 1978, p. 411. 15 P.L. Polowick, K. Raman and R.I. Greyson, Maize Genet. Coop. Newslett., 55 (1981) 116. 16 T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. 17 P. White, A Handbook of Plant Tissue Culture, J. Cattell Press, 1943. 18 P.C. Cheng, D.B. Walden and R.I. Greyson. Natl. Sci. Counc. Monthly, R. O. C., 7 (1979) 1000. 19 I.K. Vasil, Science, 126 (1957) 1294.
139
ANTHER DEVELOPMENT, MEIOSIS AND POLLEN FORMATION IN ZEA TASSELS CULTURED IN DEFINED LIQUID MEDIUM
P.L. P O L O W I C K
and R.I. G R E Y S O N
Department of Plant Sciences, Universityof Western Ontario, London, Ontario N 6 A 5B7 (Canada)
(Received October 12th, 1981) (Revision received December 15th, 1981) (Accepted January 13th, 1982)
SUMMARY
Immature tassels of corn (Zea mays L.), excised at a stage when the most mature spikelets on the main axis, possessing rudimentary stamen and gynoecial primordia, continue to grow and differentiate when cultured in a defined liquid medium. The fresh weight of the inflorescence increases up to 1000× during the 14
INTRODUCTION
Corn and tassel spikelets are initially bisexual but become unisexual through selective organ abortion [ 1 ]. It is assumed that the regulation of this differentiation involves plant growth substances [2]; however, the validity of this assumption remains unproven. Previous attempts to examine the roles of plant growth regulators in maize sexuality have involved the estimation of the endogenous levels of auxins and gibberellins [3,4] and their exogenous application [5--8]. The results, however, are contradictory and inconclusive. Floral meristems are seldom used for organogenic culture studies [9], and the completion of development, culminating in gamete maturation, is reported in few of the attempted culture studies [ 10--13 ]. Cereals, especially, have Abbreviations: FPA, formalin/propionic acid/alcohol; PMC, pollen mother cell. 0304--4211/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd.
140 been difficult to study [ 14]. The use of in vitro techniques could, potentially, aid in defining the roles of specific molecules in organ differentiation and the triggering of meiosis. As part of a broad study of sexuality in Zea mays, we have established a suitable basal medium [15] and have begun to study the effects of plant growth regulators in the liquid culture of tassels. We find that cytokinins are particularly active in tassel spikelet maturation. The observations presented in this preliminary report relate to b o t h the biochemistry of meiosis and organ differentiation. METHODS AND MATERIALS Plants of Zea m a y s (cv. Seneca-60) are raised in the greenhouse until the 8th leaf emerges and the basal internodes are elongating (approx. 25--30 days from germination). The shoot portions are removed from the pot. The t w o o u t e r m o s t leaves and the remaining exposed leaf-blades are removed to produce 10 cm stem/leaf-sheath cylinders which enclose the immature tassels. The cylinders are surface-sterilized in 15% 'Javex', sodium hypochlorite bleach, for 20 min and rinsed repeatedly in sterile water. The tassels are dissected aseptically from the stems. These tassel explants are approx. 1 cm long and bear more than 600 spikelet primordia, the most advanced of which are located in the mid-region of the central axis. The upper floret of each of these most-mature spikelets possesses b o t h stamens and gynoecium primordia, as yet undifferentiated and made up essentially of non-vacuolate meristematic cells (Fig. 1). Tassels are placed singly into 125 ml Erlenmeyer flasks which contain 40 ml of medium. The basal liquid medium, as reported previously [15], is composed of Murashige and Skoog [ 16 ] mineral salts, White's [ 17 ] vitamins and glycine, myo-inositol (Calbiochem No. 4071; 100 mg/1) and 0.1 M sucrose. Kinetin is added as indicated in the text. The pH is adjusted to 5.8 with NaOH or HC1. The medium is distributed into Erlenmeyer flasks and autoclaved. Cultures are incubated in a growth chamber on a rotary shaker (90 rev./ min) and provided with an 18-h (24°C)/6-h (20°C) photoperiod. The illumination (50 + 10 microeinsteins M -2 s -1 PAR) is provided by cool white fluorescent tubes plus incandescent bulbs. For analysis, cultures are terminated at intervals of up to 14 and occasionally 20 days. For sectioning, the cultured materials are fixed in a modified paraformaldehyde/glutaraldehyde fixative [ 18 ] or formalin/propionic acid/ alcohol (FPA) and e m b e d d e d in paraffin. For c h r o m o s o m e squashes, anthers are fixed in alcohol/glacial acetic acid (3 : 1) and stained with propiocarmine. This report summarizes observations made during an 8-month period, from over 200 cultured tassels. RESULTS AND DISCUSSION In basal medium (control), the 1-cm tassels can elongate up to 25 cm in
141
gl
O Fig. 1. Longitudinal section of a single corn floret at the time of culture showing (a) a n t h e r initials, (g) gynoecium, (gl) glume and (l) lemma. Calibration bar = 50 urn.
length and increase in fresh weight to 1500 mg from the initial 10 mg, in 14 days. Growth is evident within 36 h. A few normally-shaped and sized spikelets (~1 cm at 14 days) develop. The glumes on these spikelets are green, hairy and papery in texture, exhibit prominent venation and enclose the regular complement of lemmas, paleae and lodicules. Six 4-1oculed anthers mature, 3 for each of the t w o florets in the spikelet. Microsporogenesis proceeds to the uninucleate microspore stage. This 'normal' spikelet development is rare; a mean number of 5 spikelets developed on less than 5% o f the tassels cultured in basal medium (Fig. 2, Control}. The frequency and extent of 'normal' spikelet development is enhanced by the addition of kinetin to the medium. Kinetin is effective over a range of concentrations, 10-9--10 -~ M and is most influential at a concentration of 10 -7 M (Fig. 2). The percentage of tassels possessing 'normal' spikelets approaches 80% at this concentration and the mean number of such spikelets per tassel is nearly 18. In the most successful culture to date, a single inflor-
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escence produced 48 'normal' spikelets. This represents the development of 3--5% of the 600 ÷ initials maturing in vivo. The results are variable but t h e trend is consistent. Neither the fresh weight nor the length of the cultured tassels is enhanced by the kinetin. Similar studies with other cytokinins are in progress. There is considerable stamen growth and differentiation during the culture period. Beginning as an undifferentiated primordium (Fig. 1), the stamen matures into a 4-1oculed anther (Fig. 3) attached to a vascuiarized filament. The anthers reach 5 mm in length after 14 days, while comparable in vivo specimens are approx. 6 mm. Anatomically, cultured material resembles the in vivo-grown material with the anther wall consisting of the usual epidermal, endodermal and tapetal layers and, occasionally, a middle layer. Of particular interest is the observation that meiosis takes place in those spikelets which develop normally on cultured tassels, especially in the presence of kinetin. Vasil [19] also noted an association of kinetin with the completion of meiosis in cultured anthers of Allium cepa. Apparently, ours is the first report of the completion of microsporogenesis in a cereal inflorescence cultured when the most mature anthers are at an early initial stage. This stage is at least 3 days prior to meiosis. Undissected tassels of the same cohort of plants continue to develop in vivo for 3--5 days before pollen mother cells (PMC's) are detected. While the detailed analysis of meiosis in culture remains incomplete, it is obvious that many aspects parallel in vivo development. Within 7 days of culture, PMC's and early stages of meiotic prophase I are observed in propiocarmine squashes of anthers, as illustrated by the leptonema-zygonema stage in Fig. 4. Quartets (Fig. 5) and isolated microspores are prevalent after 10 days of culture although a range of stages is observed from individual stamens. Binucleate pollen grains (Fig. 6), bearing distinct though somewhat abbreviated walls and possessing an aperture with thickened margins, are found after 14 days of culture. Similar grains are observed after 10 days of continued growth in vivo. At this stage, the two nuclei are visibly distinct; the vegetative nucleus lies near the aperture while the generative nucleus is attached to the intine, opposite the aperture. Mature pollen, which was observed in several 20~lay-old cultures, contained a dense cytoplasm, presumably because of starch deposition. On occasion, however, stages of the mitotic divisions of the generative nucleus and the resulting trinucleate pollen grains were noted. Despite the consistency of our results with kinetin it is of concern that less than 10% of the spikelets on an inflorescence respond. Perhaps only a certain fraction is sensitive at the time of explanting. The most sensitive fraction of spikelets does not appear to be the most mature in the developmental gradient along the main and branch axes of the tassel, as the 'normal' spikelets are scattered, apparently at random along these axes. Also, the number of developing spikelets is not augmented when slightly older inflorescences are placed into culture. In view of previous experience with grass
144
Fig. 3. T r a n s v e r s e s e c t i o n o f a n a n t h e r a f t e r 10 d a y s o f c u l t u r e in m e d i u m c o n t a i n i n g 10-7 M k i n e t i n . I n d i v i d u a l locules e x h i b i t d i f f e r e n t stages o f m i c r o s p o r o g e n e s i s . Calibrat i o n bar = 50 u m . Fig. 4. Pollen m o t h e r cells at l e p t o n e m a - z y g o n e m a o f Meiosis I a f t e r 7 days of c u l t u r e in m e d i u m c o n t a i n i n g 10-7 M k i n e t i n . C a l i b r a t i o n b a r = 20 ~ m , Fig. 5. Q u a r t e t o f m i c r o s p o r e s a f t e r 10 days o f c u l t u r e in m e d i u m c o n t a i n i n g 10-7 M k i n e t i n . C a l i b r a t i o n b a r = 20 ~m. Fig. 6. B i n u c l e a t e p o l l e n grain a f t e r 14 days o f c u l t u r e in m e d i u m c o n t a i n i n g 10 -7 M k i n e t i n (a = a p e r t u r e ) . C a l i b r a t i o n b a r = 20 ~m.
145 tissues, a n y d e v e l o p m e n t in c u l t u r e is surprising, especially w i t h such a relatively simple m e d i u m . T h e s e reservations regarding n u m b e r s aside, t h e e x t e n t o f d e v e l o p m e n t f r o m s t a m e n initials t o b i n u c l e a t e pollen in 14 d a y s is striking. T h e techn i q u e s h o u l d p r o v e valuable f o r basic c y t o g e n e t i c and m o r p h o l o g i c a l studies. E x p l o r a t i o n s i n t o t h e b i o c h e m i s t r y o f meiosis and a n t h e r - d e v e l o p m e n t w o u l d be facilitated b y t h e i n c o r p o r a t i o n o f labelled-precursors, an a p p r o a c h n o t feasible in t h e i n t a c t c o r n plant. ACKNOWLEDGEMENTS We t h a n k Dr. K. R a m a n f o r his help w i t h t h e early stages o f this w o r k and gratefully a c k n o w l e d g e t h e s u p p o r t o f t h e N a t u r a l Sciences and Engineering Research Council o f C a n a d a f o r a P o s t g r a d u a t e Scholarship t o PLP a n d annual grants t o R I G . We are also grateful t o t h e University o f Western O n t a r i o A c a d e m i c D e v e l o p m e n t F u n d f o r generous s u p p o r t . REFERENCES
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